DNA and RNA are single-stranded or double-stranded molecules formed as linear chains of nucleotide bases. DNA includes the nucleotides thymine (T), cytosine (C), adenine (A) and guanine (G), and RNA is formed from T, C, G and uracil (U). In double-stranded molecules the two strands are held together by hydrogen bonding between pairs of nucleotides, with A pairing with T or U, and C pairing with G. The bases in these base pairs are called complementary bases, and the two strands are called complementary strands.
The sequence of nucleotides along a strand of RNA or DNA is relevant to many scientific studies. For example, the sequence of bases is important to analysis of genetic disease in humans and other animals, and in diagnosis of such diseases.
DNA sequencing is generally carried out by the method of Sanger et al. (74 Proc. Nat. Acad. Sci. USA 5463, 1977) and involves enzymatic synthesis of single complementary molecules of DNA from a single-stranded DNA template and a complementary primer. Briefly, referring to FIG. 1, four separate syntheses are carried out. A single-stranded template is provided along with a primer which hybridizes to the template. The primer is elongated using a DNA polymerase, and each reaction terminated at a specific nucleotide via the incorporation of an appropriate chain terminating agent, for example, a dideoxynucleotide.
The four DNA synthesis reactions result in formation of four series of DNA molecules, each molecule having one defined terminus and one variable terminus. The defined terminus starts with the primer molecule. The variable terminus ends with a chain terminating agent specific for the nucleotide (either G, A, T, or C) at which the synthesis reaction terminated. The four different series of molecules are each separated on the basis of their molecule weight, in four separate lanes in a high resolution polyacrylamide gel, to form four series of bands, with each band on the gel corresponding sequentially to a specific nucleotide in the nucleotide sequence. Thus, the relative positions of the bands identify the positions in the DNA sequence of each given nucleotide base. Generally, the DNA molecules are labelled so that the bands produced are readily detected. As shown in FIG. 1, the intensity of the bands is generally non-uniform, within a single lane, because band intensity is directly related to the total number or concentration of DNA molecules of the same molecular weight in a specific lane, and this number varies from one molecule to another even when they are of approximately the same molecular weight and even when they contain the same chain terminating agent. Tabor and Richardson, U.S. Pat. No. 4,962,020, have recently described a method for producing uniform banding, by use of a manganese buffer.
Maxam and Gilbert, 74 Proc. Nat. Acad. Sci. USA 569, 1977 describe another method for sequencing DNA where chemicals are used to specifically cleave a DNA molecule and these cleavage products analyzed by gel electrophoresis.
Fluorescent labels can be used in place of radioactive labels, as described in Fung et al., U.S. Pat. No. 4,855,225 and Hunkapiller et al. U.S. Pat. No. 4,811,218, and Prober, et al., 238 Science 336, 1987. In addition, the DNA molecules may be labeled with different isotopic variants of an atom, e.g., sulfur. The sulfur atom is used as a marker for the specific nucleotide at the end of each nucleic acid molecule, and later identified by combustion of the molecule to produce sulfur dioxide, which is then detected using mass spectrometry. Brennan, U.S. Pat. No. 5,003,059; Jacobson et al., U.S. Pat. No. 5,002,868; and Serge, EPA 0 360 676 A1.